Surgical Infection Research Group, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia.
QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia.
J Microbiol Methods. 2020 Mar;170:105856. doi: 10.1016/j.mimet.2020.105856. Epub 2020 Jan 30.
Shotgun metagenomic sequencing or metagenomic whole genome sequencing is a genome-wide sequencing approach to explore bacterial communities directly from their habitat or sites of infection. However, host DNA contamination in metagenomic sequencing overwhelm low biomass of microbial signals and decrease sensitivity for microbial detection. In this study, we evaluated the host DNA depletion efficiency of four different microbiome DNA enrichment methods (NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit and Zymo HostZERO microbial DNA kit) in diabetic foot infection (DFI) tissue samples using quantitative real-time PCR and their effect on bacterial community composition by 16S ribosomal RNA amplicon sequencing. The host DNA depletion ratio (18S/16S rRNA), the percentage of bacterial DNA component and the microbial community profile of DFI were compared before (control) and after each microbiome DNA enrichment method. There was a significant difference in the 18S/16S rRNA ratio among different microbiome DNA enrichment methods (p <.001). QIAamp and HostZERO method reduced 18S/16S rRNA ratio by 32 and 57 fold than control method respectively. The percentage of bacterial DNA component increased more than ten-fold in QiaAmp (71.0 ± 2.7%) and HostZERO (79.9 ± 3.1%) method respectively than those in control method without host DNA depletion (6.7 ± 0.1%). It demonstrated the host DNA contamination was efficiently depleted and bacterial DNA was effectively enriched in HostZERO and QIAamp methods, attesting to the efficacy of these two methods in shotgun metagenomic sequencing studies. Overall, bacterial community composition of DFI samples was similar between control and microbiome enriched DNA samples.
shotgun 宏基因组测序或宏基因组全基因组测序是一种全面的基因组测序方法，用于直接从其栖息地或感染部位探索细菌群落。然而，宏基因组测序中的宿主 DNA 污染超过了微生物信号的低生物量，并降低了微生物检测的敏感性。在这项研究中，我们使用定量实时 PCR 评估了四种不同宏基因组 DNA 富集方法（ NEBNext 宏基因组 DNA 富集试剂盒、Molzym Ultra-Deep 宏基因组预制备试剂盒、QIAamp DNA 宏基因组试剂盒和 Zymo HostZERO 微生物 DNA 试剂盒）在糖尿病足感染（DFI）组织样本中的宿主 DNA 耗竭效率，并通过 16S 核糖体 RNA 扩增子测序评估它们对细菌群落组成的影响。在每种宏基因组 DNA 富集方法之前（对照）和之后，比较 DFI 的宿主 DNA 耗竭比（18S/16S rRNA）、细菌 DNA 成分百分比和微生物群落谱。不同宏基因组 DNA 富集方法之间的 18S/16S rRNA 比值存在显著差异（p<.001）。QIAamp 和 HostZERO 方法分别将 18S/16S rRNA 比值降低了 32 倍和 57 倍，而对照方法为 16S rRNA 比值降低了 32 倍和 57 倍。QiaAmp（71.0±2.7%）和 HostZERO（79.9±3.1%）方法中的细菌 DNA 成分百分比分别比对照方法（无宿主 DNA 耗竭时为 6.7±0.1%）增加了 10 多倍。这表明 HostZERO 和 QIAamp 方法有效地耗尽了宿主 DNA 污染并有效地富集了细菌 DNA，证明了这两种方法在 shotgun 宏基因组测序研究中的有效性。总体而言，DFI 样本的细菌群落组成在对照和宏基因组富集 DNA 样本之间相似。
